Jaehee Han

Functional expression of TASK‐1/TASK‐3 heteromers in cerebellar granule cells

TASK‐1 and TASK‐3 are functional members of the tandem‐pore K+ (K2P) channel family, and mRNAs for both channels are expressed together in many brain regions. Although TASK‐1 and TASK‐3 subunits are able to form heteromers when their complementary RNAs are injected into oocytes, whether functional heteromers are present in the native tissue is not known. Using cultured cerebellar granule (CG) neurones that express mRNAs of both TASK‐1 and TASK‐3, we studied the presence of heteromers by comparing the sensitivities of cloned and native K+ channels to extracellular pH (pHo) and ruthenium red. The single‐channel conductance of TASK‐1, TASK‐3 and a tandem construct (TASK‐1/TASK‐3) expressed in COS‐7 cells were 14.2 ± 0.4, 37.8 ± 0.7 and 38.1 ± 0.7 pS (–60 mV), respectively. TASK‐3 and TASK‐1/TASK‐3 (and TASK‐3/TASK‐1) displayed nearly identical single‐channel kinetics. TASK‐3 and TASK‐1/TASK‐3 expressed in COS‐7 cells were inhibited by 26 ± 4 and 36 ± 2 %, respectively, when pHo was changed from 8.3 to 7.3. In outside‐out patches from CG neurones, the K+ channel with single channel properties similar to those of TASK‐3 was inhibited by 31 ± 7 % by the same reduction in pHo. TASK‐3 and TASK‐1/TASK‐3 expressed in COS‐7 cells were inhibited by 78 ± 7 and 3 ± 4 %, respectively, when 5 μm ruthenium red was applied to outside‐out patches. In outside‐out patches from CG neurones containing a 38 pS channel, two types of responses to ruthenium red were observed. Ruthenium red inhibited the channel activity by 77 ± 5 % in 42 % of patches (range: 72–82 %) and by 5 ± 4 % (range: 0–9 %) in 58 % of patches. When patches contained more than three 38 pS channels, the average response to ruthenium red was 47 ± 6 % inhibition (n= 5). These electrophysiological studies show that native 38 pS K+ channels of the TASK family in cultured CG neurones consist of both homomeric TASK‐3 and heteromeric TASK‐1/TASK‐3.

Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus

Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K+ channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin‐ and oxytocin‐releasing neurons. K+ channels in MNCs were identified by recording K+ channels that were open at rest in cell‐attached and inside‐out patches in symmetrical 150 mm KCl. Eight different K+ channels were identified and could be distinguished unambiguously by their single‐channel kinetics and voltage‐dependent rectification. Two K+ channels could be considered functional correlates of TASK‐1 and TASK‐3, as judged by their single‐channel kinetics and high sensitivity to pHo. Three K+ channels showed properties similar to TREK‐type tandem‐pore K+ channels (TREK‐1, TREK‐2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K+ channel was activated by application of pressure, arachidonic acid and alkaline pHi, and showed single‐channel kinetics indistinguishable from those of TRAAK. One K+ channel showed strong inward rectification and single‐channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K+ channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K+ channels. Our results show that in MNCs at rest, eight different types of K+ channels can be found and six of them belong to the tandem‐pore K+ channel family. Various physiological and pathophysiological conditions may modulate these K+ channels and regulate the excitability of MNCs.

Functional expression of TREK-2 K+ channel in cultured rat brain astrocytes.

Background K+ channels whose subunit contains four transmembrane segments and two pore-forming domains (4TM/2P) have been cloned recently. We studied whether 4TM/2P K+ channels are functionally expressed in astrocytes that are known to have a large background (resting) K+ conductance and a large resting membrane potential. Reverse transcriptase-PCR analysis showed that, among five 4TM/2P K+ channels examined, TASK-1, TASK-3 and TREK-2 mRNAs were expressed in cultured astrocytes from rat cortex. In cell-attached patches, we mainly observed three K+ channels with single-channel conductances of 30, 117 and 176 pS (-40 mV) in symmetrical 140 mM KCl. The 30 pS channel was the inward rectifying K+ channel that has been previously described in astrocytes. The 117 pS K+ channel also showed inward rectification and was insensitive to 1 mM tetraethylammonium and 1 mM 4-aminopyridine. The 176 pS channel was the Ca2+-activated K+ channel. The 117 pS K+ channel was determined to be TREK-2, as judged by its electrophysiological properties and activation by membrane stretch, free fatty acids and intracellular acidosis. In approximately 50% of astrocytes in culture, whole-cell K+ current increased markedly following application of arachidonic acid. The number of TREK-2 channels in these cells was estimated to be approximately 500-1000/cell. Our results show that TREK-2 is functionally expressed in cortical astrocytes in culture, and suggest that TREK-2 may be involved in K+ homeostasis of astrocytes during pathological states.

Expression and localization of two-pore domain K+ channels in bovine germ cells

Two-pore domain K(+) (K(2P)) channels that help set the resting membrane potential of excitable and nonexcitable cells are expressed in many kinds of cells and tissues. However, the expression of K(2P) channels has not yet been reported in bovine germ cells. In this study, we demonstrate for the first time that K(2P) channels are expressed in the reproductive organs and germ cells of Korean cattle. RT-PCR data showed that members of the K(2P) channel family, specifically KCNK3, KCNK9, KCNK2, KCNK10, and KCNK4, were expressed in the ovary, testis, oocytes, embryo, and sperm. Out of these channels, KCNK2 and KCNK4 mRNAs were abundantly expressed in the mature oocytes, eight-cell stage embryos, and blastocysts compared with immature oocytes. KCNK4 and KCNK3 were significantly increased in eight-cell stage embryos. Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed at the membrane of oocytes and blastocysts. KCNK10 and KCNK4 were strongly expressed and distributed in oocyte membranes. These channel proteins were also localized to the acrosome sperm cap. In particular, KCNK3 and KCNK4 were strongly localized to the post-acrosomal region of the sperm head and the equatorial band within the sperm head respectively. These results suggest that K(2P) channels might contribute to the background K(+) conductance of germ cells and regulate various physiological processes, such as maturation, fertilization, and development.

Comparison of Anti-Oxidant and Anti-Inflammatory Effects between Fresh and Aged Black Garlic Extracts

Numerous studies have demonstrated that aged black garlic (ABG) has strong anti-oxidant activity. Little is known however regarding the anti-inflammatory activity of ABG. This study was performed to identify and compare the anti-oxidant and anti-inflammatory effects of ABG extract (ABGE) with those of fresh raw garlic (FRG) extract (FRGE). In addition, we investigated which components are responsible for the observed effects. Hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) were used as a pro-oxidant and pro-inflammatory stressor, respectively. ABGE showed high ABTS and DPPH radical scavenging activities and low ROS generation in RAW264.7 cells compared with FRGE. However, inhibition of cyclooxygenase-2 and 5-lipooxygenase activities by FRGE was stronger than that by ABGE. FRGE reduced PGE2, NO, IL-6, IL-1β, LTD4, and LTE4 production in LPS-activated RAW264.7 cells more than did ABGE. The combination of FRGE and sugar (galactose, glucose, fructose, or sucrose), which is more abundant in ABGE than in FRGE, decreased the anti-inflammatory activity compared with FRGE. FRGE-induced inhibition of NF-κB activation and pro-inflammatory gene expression was blocked by combination with sugars. The lower anti-inflammatory activity in ABGE than FRGE could result from the presence of sugars. Our results suggest that ABGE might be helpful for the treatment of diseases mediated predominantly by ROS.

K(+) efflux through two-pore domain K(+) channels is required for mouse embryonic development.

Numerous studies have suggested that K(+) channels regulate a wide range of physiological processes in mammalian cells. However, little is known about the specific function of K(+) channels in germ cells. In this study, mouse zygotes were cultured in a medium containing K(+) channel blockers to identify the functional role of K(+) channels in mouse embryonic development. Voltage-dependent K(+) channel blockers, such as tetraethylammonium and BaCl(2), had no effect on embryonic development to the blastocyst stage, whereas K(2P) channel blockers, such as quinine, selective serotonin reuptake inhibitors (fluoxetine, paroxetine, and citalopram), gadolinium trichloride, anandamide, ruthenium red, and zinc chloride, significantly decreased blastocyst formation (P<0.05). RT-PCR data showed that members of the K(2P) channel family, specifically KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9, were expressed in mouse oocytes and embryos. In addition, their mRNA expression levels, except Kcnk3, were up-regulated by above ninefold in morula-stage embryos compared with 2-cell stage embryos (2-cells). Immunocytochemical data showed that KCNK2, KCNK10, KCNK4, KCNK3, and KCNK9 channel proteins were expressed in the membrane of oocytes, 2-cells, and blastocysts. Each siRNA injection targeted at Kcnk2, Kcnk10, Kcnk4, Kcnk3, and Kcnk9 significantly decreased blastocyst formation by ~38% compared with scrambled siRNA injection (P<0.05). The blockade of K(2P) channels acidified the intracellular pH and depolarized the membrane potential. These results suggest that K(2P) channels could improve mouse embryonic development through the modulation of gating by activators.

Verapamil Inhibits TRESK (K2P18.1) Current in Trigeminal Ganglion Neurons Independently of the Blockade of Ca2+ Influx

Tandem pore domain weak inward rectifier potassium channel (TWIK)-related spinal cord K+ (TRESK; K2P18.1) channel is the only member of the two-pore domain K+ (K2P) channel family that is activated by an increase in intracellular Ca2+ concentration ([Ca2+]i) and linked to migraines. This study was performed to identify the effect of verapamil, which is an L-type Ca2+ channel blocker and a prophylaxis for migraines, on the TRESK channel in trigeminal ganglion (TG) neurons, as well as in a heterologous system. Single-channel and whole-cell currents were recorded in TG neurons and HEK-293 cells transfected with mTRESK using patch-clamping techniques. In TG neurons, changes in [Ca2+]i were measured using the fluo-3-AM Ca2+ indicator. Verapamil, nifedipine, and NiCl2 inhibited the whole-cell currents in HEK-293 cells overexpressing mTRESK with IC50 values of 5.2, 54.3, and >100 μM, respectively. The inhibitory effect of verapamil on TRESK channel was also observed in excised patches. In TG neurons, verapamil (10 μM) inhibited TRESK channel activity by approximately 76%. The TRESK channel activity was not dependent on the presence of extracellular Ca2+. In addition, the inhibitory effect of verapamil on the TRESK channel remained despite the absence of extracellular Ca2+. These findings show that verapamil inhibits the TRESK current independently of the blockade of Ca2+ influx in TG neurons. Verapamil will be able to exert its pharmacological effects by modulating TRESK, as well as Ca2+ influx, in TG neurons in vitro. We suggest that verapamil could be used as an inhibitor for identifying TRESK channel in TG neurons.

Activation of TREK-1, but Not TREK-2, Channel by Mood Stabilizers

Earlier studies have demonstrated that the tandem pore domain weak inward rectifying K+ channel (TWIK)-related K+ (TREK)-1 channel is inhibited by antidepressants and is associated with major depression. However, little is known about the effect of mood stabilizers that are commonly used for treatment of bipolar disorder on TREK channels, members of the two-pore domain K+ (K2P) channel family. This study sought to investigate the effect of mood stabilizers on TREK-1 and TREK-2 channels. HEK-293A cells were transfected with human TREK-1 or TREK-2 DNA. The effect of mood stabilizers on TREK-1 and TREK-2 was studied using the patch clamp technique. Changes in TREK protein expression by mood stabilizers were studied in the HT-22 mouse hippocampal neuronal cells using western blot analysis. Lithium chloride (LiCl, 1 mM), gabapentin (100 μM), valproate (100 μM), and carbamazepine (100 μM) increased TREK-1 currents by 31 ± 14%, 25 ± 11%, 28 ± 12%, and 72 ± 12%, respectively, whereas they had no effect on TREK-2 channel activity. In addition, western blot analysis showed LiCl and carbamazepine slightly upregulated TREK-1 expression, but not TREK-2 in the HT-22 cells. These results suggest that TREK-1 could be a potential therapeutic target for treatment of bipolar disorders as well as depression, while TREK-2 is a target well suited for treatment of major depression.

Acceleration of Cutaneous Wound Healing by Suppression of Large Conductance Ca2+-Activated K+ Channels

Many kinds of K+ channels are involved in the regulation of cell migration and proliferation, which are required for the processes of wound healing. However, the role of K+ channels on cutaneous wound healing has not yet been reported. Here, we demonstrate that inhibition of large conductance Ca2+-activated K+ (BKCa) channels expressed in human epidermal keratinocyte facilitate cutaneous wound healing by activating both cell migration and proliferation. In the group treated with 25 mM KCl, in vivo wound healing was facilitated more rapidly than that in control group. In vitro assay of wound healing showed that 25 mM KCl significantly increased wound closure in keratinocytes after creation of linear wound with ∼200 ∈1/4m wide defect. KCl (25 mM) promoted processes of cell migration and proliferation. BKCa and two-pore domain K+ channels were recorded in the keratinocytes by using patch-clamp technique. The BKCa channel, among these K+ channels, is the most frequently observed in cell-attached mode. NS1619, a BKCa channel opener, inhibited the proliferation and migration of keratinocytes in a dose- and time-dependent manner. Charybdotoxin and iberiotoxin, BKCa channel blockers, facilitated both cell proliferation and migration by 10±7% and 30±4%, respectively. Cutaneous wound healing was also facilitated by siRNA against BKCa (BKCa/siRNA). The migration and proliferation were more enhanced by cotransfection with BKCa/siRNA and TASK-1/siRNA. BKCa channel blockers activated PKC and ERK in a time-dependent manner. These results show that BKCa and TASK-1 channels regulate proliferation and migration of human epidermal keratinocytes by activation of PKC-ERK pathway and indicate that BKCa channel could be a molecular target for regulation of cell proliferation and migration.

Dual Effect of Wogonin on TREK-2 Expression and Channel Activity

Wogonin has shown antioxidant and anti-inflammatory properties in various cell types. The aim of this study is to examine whether and how wogonin activates TREK-2, a member of the two-pore domain K+ (K2P) channel family, highly expressed in the pathological condition, such as ischemia and inflammation. Wogonin activated TREK-2 current by increasing the opening frequency. However, the wogonin-induced TREK-2 activity was decreased in a time-dependent manner, suggesting that complex signal pathway, at least two mechanisms, might be present. We first tested whether the phospholipase A2 (PLA2)- arachidonic acid (AA)-protein kinase C (PKC) signal pathways are involved in wogonin-induced TREK-2 activation. AA strongly activates TREK-2 but not TREK-2 chimera (TREK-2/TASK-3C). TREK-2/TASK-3C did not respond to application of wogonin. Wogonin failed to activate TREK-2 in the presence of PLA2 inhibitors. In the presence of PKC inhibitors, the reduction of TREK-2 activity shown after application of wogonin disappeared regardless of lapse of time. Furthermore, wogonin increased TREK-2 expression under hypoxic condition and spinal cord injury, whereas decreased the expression under nomoxic condition. These results show that wogonin has dual effect on TREK-2 channel activity and expression. Supported by R13-2005-012-01002-0 and R01-2007-000-20746-0